FR2638234A1 - BIOLOGICALLY ACTIVE OR BIOLOGICALLY ACTIVE MOLECULE CARRIER, PROCESS FOR PREPARING THE SAME AND APPLICATION THEREOF IN BIOLOGY - Google Patents

Abstract

Carrier for biological molecules, consisting of a porous inorganic matrix and of particles of a water-insoluble polymer. Biologically active carrier consisting of a porous inorganic matrix and of particles of water-insoluble polymer "sensitised" by biological molecules. They are prepared by bringing the matrix into contact with an aqueous dispersion of particles of optionally "sensitised" polymer. Use in biology.

Description

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  SUPPORT FOR BIOLOGICALLY ACTIVE MOLECULES OR HIMSELF

  BIOLOGICALLY ACTIVE, ITS PREPARATION METHOD AND

ITS APPLICATION IN BIOLOGY

  The subject of the present invention is a support for biologically active molecules or itself biologically active, its method of preparation and its application in biology (affinity chromatography,

  diagnostic tests, cell culture).

  It is known to bind biologically active molecules to polymer particles in aqueous dispersion by adsorption when it comes to polystyrene particles for example, or covalently

  when it comes to polymers having reactive groups.

  The advantage of latexes lies in the large surface area developed by the particles and in their wide range of available chemical functions. Their disadvantage, on the other hand, lies in the difficulty of using them, once atomized, as a packing system in columns or capillaries; indeed, when the polymer particles have a diameter of less than 1 micron the system

  of lining obtained lack of permeability.

  The Applicant has found a support for biologically active molecules or itself biologically active that can serve as a packing system, said system being both permeable and large.

specific surface.

  The support for biologically active molecules forming the subject of the invention is characterized in that it consists of: an inorganic, porous, non-water-soluble matrix and, on the surface of said matrix, polymer particles non-water soluble capable of binding biologically active molecules. The term "surface" means both the outer surface of the matrix and its inner surface, that is, the one developed by the pores. Among the porous inorganic materials, non-water-soluble, which can constitute the matrix, mention may be made of silicas, aluminas,

  zeolites, brick, oxides of titanium, zirconium.

  Silicas and aluminas are used on a preferential basis. The material constituting the matrix may optionally have at the surface functional groups (-COOH, -NH 2, -OH, -SH, etc.)

  capable of covalently reacting with the polymer particles.

  According to the invention, the matrix may have a pore volume of the order of 0.5 to 1.8 ml / g, a pore diameter of the order of 0.005 to 1 micron (preferably 0.1 to 1 micron} and a specific surface of

the order of 2 to 1000 m2 / g.

  It is preferably in the form of particles, especially beads having a diameter of about 4 microns to 5 mm (

preferably from 50 to 500 microns).

  The term "monomer immiscible with water" means the monomers

  having a solubility in water of less than 5% by weight.

  Among these, mention may be made of: - vinylaromatic monomers (styrene, vinyltoluene, etc.) - x-unsaturated acid alkyl esters (methyl and ethyl acrylates and methacrylates, etc.) - unsaturated carboxylic acid esters (acetate vinyl ...) - vinyl chloride; vinylidene chloride - dienes (butadiene ...) - those with nitrile functions (acrylonitrile ...)

siloxanes.

  The monomeric composition from which said polymer is derived may also contain up to 10% of its weight (preferably up to 4% of its weight) of at least one monomer carrying ionogenic or reactive groups, such as: SO3H, - OSO3H - NR3, - COOH, - OH, - NH2, - NR2,

  CH 2 CH 2 CH 2 CH 2 Cl 2 CH 2 O 3 COOR 2 PO 2

  \ O! R representing a C1 - C4 alkyl radical, preferably C1 - C: By way of example, mention may be made of: - vinylbenzene sulphonate, sulphoalkyl esters of acids

  unsaturated (2-sulfoethylmethacrylate ...).

  unsaturated carboxylic acids (acrylic acid, methacrylate)

lique, maleic, itaconic ...)

  hydroxyalkylacrylates or methacrylates (hydro-

xyethyl, hydroxypropyl ...)

  aminoalkyl esters of unsaturated acids (2-aminoethyl-

  methacrylate ... - acrylamide - vinylbenzyl chloride

glycidyl methacrylate.

  Said polymer particles may have a particle size of about 0.05 to 20 microns and preferably of the order of 0.1 to 3 microns. Most preferably, the diameter of the polymer particles is smaller than that of the pores of the matrix and

especially less than 1 micron.

  The weight of the matrix corresponds to about 1 to 1,000 times that of

  polymer particles (preferably about 50 to 500 times).

  Said support for biologically active molecules which are the subject of the invention may be prepared by "deposition" of the polymer particles on the surface of the matrix by simple contact of an aqueous dispersion (latex) of polymer particles with the porous matrix then drying after washing (with distilled water). The latex used contains approximately 0.1 to 50% (preferably 0.5 to 15%) by weight of

polymer particles.

  The relative amounts of matrix and aqueous dispersion used correspond to a weight ratio matrix / polymer particles of the order of 0.01 to 1000; depending on the concentration of the dispersion and the physical characteristics of the matrix, the amount of particles used corresponds to about 1 to 100 times that deposited. The non-useful dispersion can then be reworked after

  separation of the obtained support, in a subsequent operation.

  When said support is prepared from a matrix having at the surface functional groups capable of covalently reacting with the polymer particles, it is preferable to preactivate the matrix with the aid of a coupling agent (carbodiimide-water-soluble, glutaraldehyde, N-hydroxybenzotriazole, cyanogen bromide, etc.) and then bring it into contact with the polymer dispersion. Said support described above can be used in biology to immobilize biologically active substances (proteins such as antibodies, enzymes, antigens, drugs, etc.) by adsorption or covalence; depending on the nature of said active substance, it is possible to obtain a support which can advantageously be used in diagnostic tests (agglutination "RIA" radioimmunological assay - "IRMA" immunoradiometric assay - "EIA" enzyme immunoassay), in affinity chromatography as an enzymatic catalyst in

  biotechnology, as a cell culture support.

  The subject of the present invention is also the biologically active supports consisting of: an inorganic, porous non-water-soluble matrix and, on the surface of said matrix, non-water-soluble polymer particles "sensitized" by an effective quantity of substances

biologically active.

  The amount of "sensitizing" substance present is generally of the order of 0.01 to 15% by weight relative to the weight of the polymer particles. It is obvious that this quantity is chosen in

  depending on the desired application of the biologically active medium.

  By polymer particles "sensitized" by biologically active substances, polymer particles on

  which said substances are fixed by adsorption or covalence.

  The nature and characteristics of the matrix as well as the nature and characteristics of the polymer particles have already been given

above.

  Among the "sensitizing" biologically active substances, mention may be made of proteins (antibodies, enzymes, etc.), antigens, drugs, etc. Said biologically active carriers which are the subject of the invention may be prepared by "depot" polymer particles "sensitized" by biologically active substances on the surface of the matrix by simply contacting an aqueous dispersion (latex) of polymer particles "sensitized" with biologically active substances, with the porous matrix, and then drying after washing possible, at a temperature compatible with the substance

  biologically active fixed on said particles.

  The "sensitization" of the polymer particles by the biologically active substance can be carried out by adsorption or by coupling, the coupling reaction involving the reactive functional groups of the polymer and the functional groups of the biological molecules. The coupling reaction can be carried out according to well-known methods, for example by using coupling agents (such as glutaraldehyde, water-soluble carbodiimide, N-hydroxybenzotriazole spacers of the 1-6 diaminohexane type, polysaccharide, etc.) by activation. functions of the polymer (for example by diazotization, by the action of cyanogen bromide, tosyl chloride, etc.)

  then reaction with the molecule to be fixed.

15. etc ...

  Examples of coupling reactions are given in US Pat. Nos. 3,857,931, 4,045,384, 4,140,662, 4,046,723, 4,421,896, English No. 2,004,892, French No. 2,331,567, No. 2,345,459, 2,378,094,

European No 15,841 ...

  An alternative embodiment of the method for preparing said biologically active supports consists of "depositing" non-sensitized polymer particles on the surface of the matrix according to the method previously described for the supports for biologically active molecules, and then to fix the molecules biologically. active on the polymer particles by adsorption or coupling according to

the methods described above.

  The biologically active supports which are the subject of the invention may be used, depending on the nature of the active substance immobilized on the polymer particles, in diagnostic tests ("RIA" radioimmunological assay - "IRMA" immunoradiometric assay - "EIA "enzyme immunoassay"), in affinity chromatography, as an enzymatic catalyst in biotechnology, as a support for

cellular culture.

  They are particularly useful as diagnostic tests when the immobilized active substance is an immunoreactant (antigen or antibody); in particulate form, said supports can constitute the packing of columns and in particular of capillary tubes that can be used for carrying out qualitative or semi-quantitative tests. The following examples are given for information only and can not be considered as a limit of the scope and spirit of the invention.

EXAMPLE

  Preparation of a "Sensitized" Polymer Particle Latex F (ab ') 2 Anti protein C rabbit antibody fragments are incubated at a concentration of 1 mg / l in phosphate buffered saline at pH 7.8 the presence of an ESTAPOR K 025 latex sold by RHONEPOULENC (calibrated latex consisting of 0.25 micron diameter polystyrene microspheres) at a concentration of 1% by weight of

  polymer particles in the buffer.

  After 17 hours of incubation at 22 ° C., the sensitized latex is washed three times with water, centrifuged and then redispersed in phosphate gelatin buffer at a concentration of 1% by weight of

polymer in the buffer.

  Deposition of polymer particles "sensitized" on a porous silica The dispersion of "sensitized" polymer particles is diluted

five times with distilled water.

  Fine rain and with gentle stirring of silica SPHEROSIL XOC 005 marketed by RHONE-POULENC (silica grains 100 to 200 microns in diameter, having a pore volume of 1 ml / g, a pore diameter of 0, are added to it. 30 micron and a specific surface area of m 2 / g), at a rate of 10% by weight relative to the dispersion weight

  (ie 1000% by weight relative to the weight of the particles).

  After two hours of impregnation at rest and at room temperature, the silica is washed five times with distilled water and then dried in an oven

at 501C for two hours.

  Making a capillary test A PASTEUR pipette type capillary tube is closed at its lower end by a hydrophilic cotton pad. The capillary portion of the tube is filled under vibration using a stirrer of the type REAX 2000 marketed by Prolabo, silica previously

  prepared, on a height of 5 cm.

  The upper end of the capillary tube is closed with a buffer of

cotton wool.

  Qualitative diagnostic test (sandwich technique) The lower end of the capillary tube is immersed in a

  aqueous solution of protein C (antigen).

  The silica column is filled in less than 5 minutes. The tube is incubated for 2 hours at room temperature. The tube, with a suction dropper is immersed in a washing solution

  (TWEEN phosphate buffer) and inverted to remove wash water.

  The tube, with its dropper dispersion is then immersed in an aqueous solution of antiprotein C-peroxidase antibody until

total impregnation of the support.

  After incubation for two hours at room temperature and then washing as above, a solution of ABTS substrate (based on 2-2'-azinodi3 ethylbenzthiazoline sulfate) reveals the presence of

  Protein C by staining the silica in blue-green.

Claims (14)

  1 Support for biologically active molecules characterized in that it consists of: - an inorganic, porous, non-water-soluble matrix, and on the surface of said matrix, non-water-soluble polymer particles capable of binding biologically active molecules. 2 support according to claim 1, characterized in that said matrix has a pore volume of the order of 0.5 & 1.8 ml / g, a pore diameter of about 0.005 to 1 micron, and a surface   specificity of the order of 2 to 1000m2 / g.   3 support according to claim 1 or 2, characterized in that said matrix is in the form of particles of diameter of the order from 4 microns to 5 mm.   4 Support according to any one of the preceding claims,   characterized in that the material constituting the matrix is of the silica, alumina.   Support according to any one of the preceding claims,   characterized in that the polymer particles have a   particle size on the order of 0.05 to 20 microns.   6 Support according to claim 5, characterized in that the diameter of the polymer particles is smaller than that of the pores of the matrix.   Support according to any one of the preceding claims,   characterized in that the weight of the matrix is about 1   at 1000 times that of the polymer particles.   Support according to any one of the preceding claims,   characterized in that the water-insoluble polymer is derived from a vinylaromatic monomer, an unsaturated acid alkyl ester, an ester of an unsaturated carboxylic acid, vinyl chloride, vinylidene chloride, a conjugated diene of an unsaturated monomer   having a nitrile function, a siloxane.   Support according to any one of the preceding claims,   characterized in that the water-insoluble polymer is derived from a water immiscible monomer and less than 10% from at least one   comonomer carrying an ionogenic or reactive group.   Support according to Claim 9, characterized in that the comonomer is divinylbenzene sulphonate, an unsaturated acid sulphoalkyl ester, an unsaturated carboxylic acid, a hydroxyalkylacrylate or methacrylate, an unsaturated acid aminoalkyl ester, acrylamide, vinylbenzene chloride, methacrylate of glycidyl.   11Support according to any one of the preceding claims,   characterized in that the matrix has at the surface functional groups capable of covalently reacting with the polymer particles.   12 Process for preparing the support subject of the one   any of the preceding claims by deposition of particles of   non-water-soluble polymer capable of binding biologically active molecules to the surface of an inorganic, porous, non-water-soluble matrix by contact with an aqueous dispersion   said polymer particles with the porous matrix and drying.   Process according to Claim 12, characterized in that the dispersion of polymer particles contains from 0.1 to 50% by weight of polymer particles.   Process according to Claim 12 or 13, characterized in that the relative amounts of matrix and aqueous dispersion used correspond to a matrix / particle weight ratio of   polymer of the order of 0.01 to 1000.   A biologically active carrier formed of the carrier for biologically active molecules that is the subject of any of the   claims 1 & 11, the polymer of which is "sensitized" by biologically active molecules.   Biologically active carrier according to claim 15, characterized in that the biologically active "sensitizing" substances   are proteins, antigens, drugs.   Bi-logically active carrier according to claim 15 or 16, characterized in that the amount of "sensitizing" substance of the polymer is on the order of 0.01 to 15% by weight relative to   the weight of polymer particles.   18 Process for preparing the biologically active carrier making   the subject matter of any one of claims 15 to 17 by fixation   of biologically active molecules on the support being the object   of any one of claims 1 to 11.   19. Process according to claim 18, characterized in that the quantity of biologically active molecules used is sufficient.   to sensitize from 0.01 to 15% of the weight of polymer.   Use of the support for the purpose of any of the   Claims 1 to 11 and 15 to 17 in biology.   21 Diagnostic reagent constituted by the biologically active carrier   subject of any one of claims 15 to 17.